CN117205235A - Microcapsule bacterial mixture for improving compulsive behavior as well as preparation method and application thereof - Google Patents

Abstract

The application discloses a microcapsule bacterial mixture for improving compulsive behaviors, a preparation method and application thereof, which belong to the technical field of medicines, after mixed bacterial liquid of two bacteria of Acremonium and F.PB1 bacteria is uniformly mixed with sodium alginate, calcium alginate gel is formed by dripping calcium chloride solution at a constant speed under the action of an extrusion device, and the microcapsule bacterial mixture with a three-layer structure is further formed by wrapping chitosan and acacia. The application provides a practical method for providing a powerful theoretical basis and bacterial liquid as medicine application in seeking the treatment potential of probiotics on obsessive-compulsive disorder, and has higher social significance and potential market value.

Description

Microcapsule bacterial mixture for improving compulsive behavior as well as preparation method and application thereof

Technical Field

The application belongs to the technical field of medicines, and particularly relates to a microcapsule bacterial mixture for improving compulsive behaviors, and a preparation method and application thereof.

Background

Obsessive-compulsive-compulsive disorder (OCD) is a chronic, disabling, common mental disorder characterized by recurrent invasive compulsive thinking and compulsive behavior, and has become the fourth most common mental disorder disease following depression, alcohol/drug abuse and social phobia, affecting about 1-4% of the population worldwide. The lifetime prevalence of obsessive-compulsive disorder is 2-3%, there is no obvious difference in prevalence between gender, race and socioeconomic class, most cases are between 12-21 years old, and usually last for decades, with the estimated annual required social costs exceeding $80 billion. Once the disease is severe, it is difficult to self-relieve, and intervention by external factors is often required to relieve the symptoms.

Psychological treatments are recommended by the national health and wellness medical research center (National institute for health and care excellence, NICE) as the treatment of choice for pediatric and adolescent compulsive patients, including cognitive behavioral therapy (Cognitive behavior therapy, CBT) and response prevention exposure (Exposure with response prevention, ERP), which repeatedly expose compulsive patients to terrible stimuli while requiring them to avoid compulsive behavior. While psychotherapy is effective in reducing compulsive thinking and compulsive behavior, about 50% of patients are in conflict with such treatment due to lack of motivation and participation, and therefore, are often in need of drug-co-therapy. Selective 5-hydroxytryptamine reuptake inhibitors (Serotonin-selective reuptake inhibitors, SSRIs) and 5-hydroxytryptamine-norepinephrine reuptake inhibitors (Serotonin-norepinephrine reuptake inhibitors, SNRIs) are first line treatment drugs for obsessive compulsive disorder, with over 90% of patients taking drugs over a long period of time. Common SSRIs drugs, such as Citalopram (Citalopram), fluoxetine (Fluvoxamine), fluvoxamine (Fluvoxamine), paroxetine (Paroxyxetine) and Sertraline (Sertraline), have therapeutic effects on both the acute and maintenance phases of obsessive-compulsive disorder, and there is no significant difference between the therapeutic effects of the drugs. Clinical practice often uses SSRIs in combination with CBT or ERP to obtain better efficacy. Even so, 40-60% of patients remain insensitive to drug treatment and require other deep treatment modalities. Neuromodulation is the last means of treating obsessive-compulsive disorder, and current is typically used to stimulate brain regions to correct electrical brain abnormalities, and clinical treatment has been shown to improve the yarrowia-Brown obsessive-compulsive scale (Y-BOCS) score of obsessive-compulsive patients. Nevertheless, some patients still have residual symptoms after undergoing various treatments. Therefore, it is particularly important to find other alternatives.

Disclosure of Invention

In order to solve the technical problems, the application provides a microcapsule bacterial mixture for improving compulsive behaviors, and a preparation method and application thereof.

In order to achieve the aim, the application provides a microcapsule bacterial mixture for improving compulsive behaviors, which has a core-shell structure, wherein the outer layer is Arabic gum, the middle layer is chitosan, and the inner layer is a mixed solution of the bacterial mixture and sodium alginate;

the bacteria in the bacterial mixture are Acremonium and F.PB1 bacteria.

The preparation method of the microcapsule bacterial mixture for improving the compulsive behavior comprises the following steps:

step 1: collecting Ackermans bacterial liquid and F.PB1 bacterial liquid which grow to a plateau, respectively centrifuging, discarding the supernatant, re-suspending the bacterial bodies by using sterile PBS, respectively centrifuging, discarding the supernatant, repeatedly washing for three times, mixing, re-suspending the bacterial bodies by using sterile PBS containing 25% glycerol, and regulating the OD600 of the bacterial liquid to be 1.2;

step 2: 2-10 mL of the bacterial liquid in the step 1 is taken and added into sodium alginate solution according to the volume ratio of 1:1, and magnetically stirred to obtain solution;

step 3: extruding the solution obtained in the step 2 into 0.1M CaCl at constant speed ₂ Forming calcium alginate gel in the solution, hardening the gel at 4deg.C for 1 hr, removing supernatant, washing the alginic acid with sodium chloride solutionA calcium gel;

step 4: immersing the calcium alginate gel into a chitosan water solution, uniformly stirring to obtain calcium alginate/chitosan gel, washing the calcium alginate/chitosan gel with a NaCl solution, and standing;

step 5: immersing the calcium alginate/chitosan gel in the step 4 in a 1wt% acacia solution, stirring and coating for 30min, and filtering and recovering the microcapsule bacterial mixture for improving the compulsive behavior.

After mixed bacterial liquid of two bacteria (hereinafter referred to as A+F mixed bacterial liquid) of Acremonium and F.PB1 bacteria is uniformly mixed with sodium alginate, the mixed bacterial liquid is dropped into a calcium chloride solution at uniform speed under the action of an extrusion device to form calcium alginate gel, and the calcium alginate gel is further wrapped by chitosan and Arabic gum to form a microcapsule bacterial mixture with a three-layer structure, so that the microcapsule has obvious improvement effect on the compulsory sample behavior of mice, and is beneficial to the preservation and colon release of the bacterial liquid.

Preferably, the effective dosages of the Ackermans bacteria solution, the F.PB1 bacteria solution and the A+F mixed bacteria solution are all 200 mu L, and the concentrations of the Ackermans bacteria solution, the F.PB1 bacteria solution and the A+F mixed bacteria solution are all controlled to be 1.2x10 ⁹ CFU。

Preferably, in step 1, the temperature is 4 ℃ and the rotation speed is 4000rpm during two times of centrifugation, and the time is 10min.

Preferably, in the step 2, the magnetic stirring time is 30-60 min, and the rotating speed is 200 rpm.

Preferably, the preparation method of the microcapsule bacterial mixture for improving compulsive behavior specifically comprises the following steps:

step 1: collecting Ackermans bacterial liquid and F.PB1 bacterial liquid growing to a plateau, respectively centrifuging for 10min at 4 ℃ and 4000rpm by using a refrigerated centrifuge, discarding the supernatant, re-suspending bacterial bodies in an anaerobic incubator by using sterile PBS, centrifuging for 10min at 4 ℃ and 4000rpm, discarding the supernatant, repeatedly washing for three times, mixing, re-suspending bacterial bodies by using sterile PBS containing 25% (volume fraction) glycerol, adjusting the OD600 of the bacterial liquid to be 1.2, and storing at-80 ℃ for standby;

step 2: 2-10 mL of the bacterial liquid in the step 1 is taken and added into sodium alginate solution with the concentration of 2wt% according to the volume ratio of 1:1, and magnetically stirred for 30-60 min, and the rotating speed is 200 rpm, so as to obtain solution;

step 3: connecting the solution obtained in the step 2 with a chip through a pressure pump, and accessing the chip into an extrusion device, and extruding into 0.1MCaCl at a constant speed ₂ Forming calcium alginate gel in the solution, and forming CaCl at the bottom of the nozzle ₂ The distance between the surfaces of the solutions was 3cm, the gel was allowed to harden at 4℃for 1h, and the supernatant was removed, and the calcium alginate gel was washed twice with an autoclaved 0.85wt% sodium chloride (NaCl) solution;

step 4: 0.4wt% chitosan aqueous solution was prepared: 0.4g of chitosan was dissolved in 90mL of distilled water and 0.4mL of glacial acetic acid, the pH was adjusted to 6.0 with NaOH, the total volume was adjusted to 100mL with ultrapure water, and the solution was autoclaved and filtered to remove solids that were not completely dissolved, yielding a 0.4wt% aqueous chitosan solution. Immersing the calcium alginate gel in the step 3 into the chitosan water solution, stirring for 10min to obtain calcium alginate/chitosan gel (Al/Chi gel), rinsing the Al/Chi gel twice with sterilized 0.85wt% NaCl solution, and standing for 1h at 4 ℃;

step 5: immersing the Al/Chi gel in the step 4 in a 1wt% acacia solution, stirring and coating for 30min, and filtering and recovering the microcapsule bacterial mixture (which is a three-layer microcapsule structure of bacterial mixture/calcium alginate-chitosan-acacia) for improving the compulsory behavior.

The application of the microcapsule bacterial mixture for improving compulsive behavior in preparing medicines for treating compulsive disorder.

Preferably, the obsessive-compulsive disorder includes, but is not limited to, obsessive-compulsive disorder caused by immune dysfunction of the intestinal tract.

Preferably, the dosage form of the medicament comprises any pharmaceutically acceptable dosage form.

Preferably, the formulation of the drug includes any one of a liquid formulation, a solid formulation, a semisolid formulation and a gaseous formulation.

Preferably, the mode of administration of the forced behavior-improving micro-encapsulated bacterial mixture is intragastric administration.

Compared with the prior art, the application has the following advantages and technical effects:

1) The application provides a new application of Acremonium and F.PB1 bacteria in preparing a microcapsule bacterial preparation for treating obsessive-compulsive disorder, which can regulate synthesis of neurotransmitter Dopamine (DA) and dysfunction of intestinal barrier by balancing intestinal microecology, thereby greatly relieving abnormal psychological behavior state related to obsessive-compulsive disorder. The application verifies that the microcapsule bacterial mixture prepared by mixing Acremonium and F.PB1 bacteria improves the secretion of neurotransmitter DA and the expression of Th, thereby improving the related symptoms of obsessive compulsive disorder.

2) The application selects C57BL/6J healthy male mice with the age of 8 weeks and the weight of 20-22g, adopts the subcutaneous injection of 1mg/kg QNP from the cervical back, and the injection is carried out once every three days, and the control group is given with the same amount of physiological saline for 10 times, thus constructing the sub-chronic compulsive behavior animal model. After the gastric lavage PBS, the Ackermans, the F.PB1 bacteria and the A+F mixed bacteria liquid are respectively carried out for 14 days, the forced sample behavior and the anxiety behavior of the A+F mixed bacteria liquid group are observed to be improved most remarkably compared with other groups, the synthesis of neurotransmitter dopamine is also obviously up-regulated, and the effects are observed to be more obvious after the A+F mixed bacteria liquid is subjected to microcapsule packaging. From the above experimental results, it is presumed that the microcapsule bacterial mixture made of ackermanni and f.pb1 bacteria may alleviate obsessive compulsive symptoms by regulating dopamine synthesis. The application provides a powerful theoretical basis for seeking the treatment potential of probiotics on obsessive-compulsive disorder, and has higher social significance and potential market value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

FIG. 1 is a schematic structural diagram of a microencapsulated bacterial cocktail that improves forcing behavior;

FIG. 2 is a view of an image under a microscope of the present application;

FIG. 3 shows the forced behavior results of a model mouse induced by the intervention QNP of the mixed bacterial liquid of Ackermans and F.PB1, wherein A is an open field experiment, B is an open field experiment trace diagram, C is an elevated plus maze experiment trace diagram, E is a marble burying experiment, and F is a Y maze experiment;

FIG. 4 shows the compulsory behavior results of a model mouse induced by the intervention QNP of the microcapsule Acremonium and F.PB1 mixed bacterial liquid, wherein A is an open field experiment, B is an open field experiment trace diagram, C is an elevated plus maze experiment trace diagram, E is a marble burying experiment, and F is a Y maze experiment;

FIG. 5 shows the effect of the mixed bacterial liquid of Acremonium and F.PB1 on the synthesis of neurotransmitter DA in the cortex of a model mouse, wherein A is a Western Blot experiment for detecting the expression of Th proteins in the cortex of the mouse, B is a qPCR experiment for detecting the expression of Th genes in the cortex of the mouse, and C is a high performance liquid chromatography experiment for detecting the DA content in the cortex of the mouse;

FIG. 6 shows the effect of mixed bacterial liquid intervention of microcapsule Acremonium and F.PB1 on DA synthesis of a model mouse cortex neurotransmitter, wherein A is a Western Blot experiment for detecting expression of Th protein in the mouse cortex, B is a qPCR experiment for detecting expression of Th gene in the mouse cortex, and C is a high performance liquid chromatography experiment for detecting DA content in the mouse cortex;

FIG. 7 shows the survival rate of the microencapsulated bacterial cocktail in simulated gastric fluid to improve compulsive behaviour;

figure 8 shows the survival rate of the microencapsulated bacterial cocktail in simulated intestinal fluid to improve compulsive behaviour.

Detailed Description

Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

Acremonium in the examples of the present application was deposited under the accession number ATCC BAA-835, available from American type culture Collection; the F.PB1 strain has a accession number JCM 30274, which is purchased from the department of physical and chemical research of Japan biological resource center.

The experimental methods used in the embodiments of the present application are all conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In the embodiment of the application, the bacteria in the Akk group and the F.PB1 group grow to the stage.

PBS phosphate buffer (dry powder) in the examples of the present application was purchased from biosharp corporation at a concentration of 0.01M.

Example 1

A preparation method of a microcapsule bacterial mixture for improving compulsive behavior comprises the following steps:

step 1: collecting Ackermans bacterial liquid and F.PB1 bacterial liquid growing to a plateau, respectively centrifuging for 10min at 4 ℃ and 4000rpm by using a refrigerated centrifuge, discarding the supernatant, re-suspending bacterial bodies in an anaerobic incubator by using sterile PBS, centrifuging for 10min at 4 ℃ and 4000rpm, discarding the supernatant, repeatedly washing for three times, mixing, re-suspending bacterial bodies by using sterile PBS containing 25% (volume fraction) glycerol, adjusting the OD600 of the bacterial liquid to be 1.2, and storing at-80 ℃ for standby;

step 2: adding 6mL of the bacterial liquid in the step 1 into a sodium alginate solution with the concentration of 2wt% according to the volume ratio of 1:1, magnetically stirring for 45min, and obtaining a solution at the rotating speed of 200 rpm;

step 3: connecting the solution obtained in the step 2 with a chip through a pressure pump, and accessing the chip into an extrusion device, and extruding into 0.1MCaCl at a constant speed ₂ Forming calcium alginate gel in the solution, and forming CaCl at the bottom of the nozzle ₂ The distance between the surfaces of the solutions was 3cm, the gel was allowed to harden at 4℃for 1h, and the supernatant was removed, and the calcium alginate gel was washed twice with an autoclaved 0.85wt% NaCl solution;

step 4: 0.4wt% chitosan aqueous solution was prepared: 0.4g of chitosan was dissolved in 90mL of distilled water and 0.4mL of glacial acetic acid, the pH was adjusted to 6.0 with NaOH, the total volume was adjusted to 100mL with ultrapure water, and the solution was autoclaved and filtered to remove solids that were not completely dissolved, yielding a 0.4wt% aqueous chitosan solution. Immersing the calcium alginate gel in the step 3 into the chitosan water solution, stirring for 10min to obtain Al/Chi gel, rinsing the Al/Chi gel twice with sterilized 0.85wt% NaCl solution, and standing for 1h at 4 ℃;

step 5: immersing the Al/Chi gel in the 1wt% acacia solution, stirring and coating for 30min, filtering and recovering to obtain the microcapsule bacterial mixture for improving the compulsory behavior, wherein the structural schematic diagram of the microcapsule bacterial mixture is shown in figure 1, the microscopic image is shown in figure 2, and the microcapsule bacterial mixture is a three-layer microcapsule structure of the bacterial mixture/calcium alginate-chitosan-acacia (the outer layer is acacia, the middle layer is chitosan, and the inner layer is a mixed solution of the bacterial mixture and sodium alginate).

A preparation method of microcapsule PBS for improving compulsive behavior comprises the following steps:

step 1: adding 6mL of sterile PBS containing 25% (volume fraction) glycerol into sodium alginate solution with concentration of 2wt% according to the volume ratio of 1:1, magnetically stirring for 45min, and rotating at 200 rpm to obtain a solution;

step 2: connecting the solution obtained in the step 1 with a chip through a pressure pump, and accessing the chip into an extrusion device, and extruding into 0.1MCaCl at a constant speed ₂ Forming calcium alginate gel in the solution, and forming CaCl at the bottom of the nozzle ₂ The distance between the surfaces of the solutions was 3cm, the gel was allowed to harden at 4℃for 1h, and the supernatant was removed, and the calcium alginate gel was washed twice with an autoclaved 0.85wt% NaCl solution;

step 3: 0.4wt% chitosan aqueous solution was prepared: 0.4g of chitosan was dissolved in 90mL of distilled water and 0.4mL of glacial acetic acid, the pH was adjusted to 6.0 with NaOH, the total volume was adjusted to 100mL with ultrapure water, and the solution was autoclaved and filtered to remove solids that were not completely dissolved, yielding a 0.4wt% aqueous chitosan solution. Immersing the calcium alginate gel obtained in the step 2 into the chitosan water solution, stirring for 10min to obtain Al/Chi gel, rinsing the Al/Chi gel twice with sterilized 0.85wt% NaCl solution, and standing for 1h at 4 ℃;

step 4: and (3) immersing the Al/Chi gel in the step (3) in a 1wt% Arabic gum solution, stirring and coating for 30min, and filtering and recovering to obtain the microcapsule PBS mixture.

EXAMPLE 2 Performance experiments on forced behavior-improving microencapsulated bacterial cocktail

The experimental animals selected were:

c57BL/6J healthy male mice with the age of 8 weeks and the weight of 20-22g are selected, the room temperature is kept at 23+/-1 ℃, and the humidity is kept at 50+/-10%. The light and dark cycle is carried out for 12 hours, and clean food and water can be obtained at will.

Experimental method 1: experimental animals were randomly divided into four groups, PBS group (PBS), ackermannia group (Akk), f.pb1 group (f.pb1), ackermannia and f.pb1 mixed bacteria liquid group (a+f). Four groups of mice were injected with 10 injections of quepirole (Quinpirole, QNP) for the establishment of a compulsive disorder model, and 200 μl of PBS (PBS group) and 1.2X10 concentrations were respectively injected once daily for two weeks after modeling ⁹ 200. Mu.L of Ackermans bacteria liquid of CFU (Akk group), 200. Mu.L of F.PB1 bacteria liquid (F.PB1 group), 200. Mu. L A +F mixed bacteria liquid (A+F group). The daily administration time is8 a.m.: 00-9:00, continuously administering for 14d, and performing a behavioural experiment on each group of mice on day 15 for 12h on an empty stomach; on day 16, mice in each group were fasted for 12 hours, blood was collected from the orbit, centrifuged at 3000r/min, and serum was retained for use. The brain tissue (cortex) and colon are used for detection by Western blot method, qPCR method, HPLC method, etc. All charts and analyses were performed by GraphPad Prism 8.0, data were expressed as mean ± standard error (mean ± s.e.m), and statistical significance (P) was assessed using a common one-way analysis of variance (Analysis of variance, ANOVA) and a minimum significant difference (Least significant difference, LSD) multiple test<0.05)，*P<0.05，**P<0.01，***P<0.001。

Experimental method 2: the experimental animals are randomly divided into four groups, namely PBS group (PBS), microcapsule PBS group (MC-PBS), A+F mixed bacterial liquid group (A+F) and microcapsule A+F mixed bacterial liquid group (MC-A+F). Four groups of mice were injected with quepirole (Quinpirole, QNP) 10 times for constructing compulsive disorder models, and 200 μl or equal amounts of PBS, microcapsule PBS, a+f mixed bacterial liquid, and microcapsule a+f mixed bacterial liquid (i.e., the microcapsule bacterial mixture for improving compulsive behavior prepared in example 1) were respectively injected once daily for two weeks after modeling. Daily dosing time was 8 a.m.: 00-9:00, continuously administering for 14d, and performing a behavioural experiment on each group of mice on day 15 for 12h on an empty stomach; on day 16, mice in each group were fasted for 12 hours, blood was collected from the orbit, centrifuged at 3000r/min, and serum was retained for use. The brain tissue (cortex) and colon are used for detection by Western blot method, qPCR method, HPLC method, etc. All charts and analyses were performed with GraphPad Prism 8.0, data expressed as mean ± standard error (mean ± s.e.m), and statistical significance (P < 0.05), P <0.01, P <0.001 was assessed using a common one-way analysis of variance (Analysis of variance, ANOVA) and a minimum significant difference (Least significant difference, LSD) multiple test.

(1) Effect of bacterial fluid and microcapsule packaging on compulsive behavior of QNP-induced model mice

The daily dosing time for each of the above groups of mice was 8 a.m.: 00-9:00, continuously administering 14d, and carrying out a behavioral experiment on each group of mice of 15d for 12h on an empty stomach, wherein the specific method is as follows:

(1) ball burying experiment

A Marble Burying (MB) experiment has been used to evaluate the compulsory behavior of animals, the experiment being performed following protocols described by Deacon et al. Briefly, a clean cage with a length, width and height of 40×24×16cm was prepared, wood chip padding with a thickness of 5cm was laid on the bottom of the cage, and 18 glass marbles with a diameter of 1.5cm were placed on the surface of the padding, and were arranged at equal intervals in 6 rows×3 columns. The mice were placed individually in the center of the cage, the cage was freely explored under dim lighting conditions without interference, and after 15min, the mice were removed and the number of marbles with a burial depth exceeding 2/3 was calculated. The greater the number of burial of the marbles indicates a greater forced burial behavior, the results being expressed as a percentage of the number of burial of the marbles.

(2) Y maze

The reduction in spontaneous alternating behavior in rodents has been proposed as an animal model of persistent symptoms in obsessive-compulsive patients, and the detection has been performed according to previously reported procedures. Specifically, in a Y maze (Y maze) with an arm length of 30cm, a width of 8cm and a height of 15cm, one arm is taken as a starting arm, a mouse is placed at the tail end of the starting arm facing the selection arm, the mouse can freely select to enter a left side arm or a right side arm, the selected arm is recorded as a target arm, and the mouse is allowed to freely move in the target arm for 5min. The mice were then replaced at the end of the starting arm, observed for entry into the target arm, repeated 20 times, and the number of times the mice entered the target arm was recorded. The more target arm entries indicate more forced repetitive behavior, and the results are expressed as a percentage of the target arm entries.

(3) Open field experiment

Overchecking is a common, debilitating symptom OF obsessive-compulsive patients, and animals can be simulated for compulsive checking with a common Open field facility (OF). Dividing a 50 x 50cm open field into a grid of 25 virtual rectangular areas, but without any actual marking lines on the open field surface, and simultaneously fixing four 4 x 4cm objects on the open field surface: two are located at the corners and two are located near the center. Each mouse was placed individually in the center of the open field and allowed to explore freely for 15min. The track of the mice was automatically tracked using a camera tracking system. Mice were considered to have forced examination behavior when they accessed 1-2 of them too frequently and quickly.

(4) Overhead cross maze

Obsessive compulsive patients often co-suffer from anxiety disorders and elevated plus maze (Elevated plus maze, EPM) are used to test anxiety-related behavior in rodents. The EPM device was placed at a distance of 50cm from the ground and 1m from the wall, with the arm length of 30cm, width of 5cm and height of 15cm. The mice were placed in an EPM device facing the open arm and were freely explored for 5min, and the track of each animal was automatically tracked using a camera tracking system. The reduction in distance and duration of movement in the open arms and the number of open arms entered reflects anxiety-like behavior.

The above behavioural results are shown in fig. 3 and 4, in fig. 3, the AKK, f.pb1, a+f mice all showed a significant reduction in forced burying behavior, forced repetitive behavior, forced examination behavior and anxiety-like behavior, and the a+f mice showed the most significant differences compared to the PBS mice, while the other behavioural experimental indexes described above were not significantly changed. The result shows that the interference of Acremonium and F.PB1 bacteria can improve the forced behavior of a model mouse induced by QNP, and the mixed bacterial liquid of A and F can achieve the most obvious effect. In fig. 4, both the a+f group and the MC-a+f group showed significant compulsory burying behavior, compulsory repeating behavior, compulsory checking behavior, and anxiety-like behavior alleviation compared to the PBS group mice, and the MC-a+f group showed more significant differences, not significantly compared to the PBS group. The microcapsule package is shown to improve the capacity of the A+F mixed bacterial liquid to intervene and improve the forced behavior of a model mouse induced by QNP, and the microcapsule PBS package has no obvious influence on the behavior of the mouse compared with the PBS group.

(2) Influence of bacterial sap and microcapsule packaging intervention on model mouse cortical neurotransmitter DA synthesis

Experimental method 1: experimental animals were randomly divided into four groups, PBS group (PBS), ackermannia group (Akk), f.pb1 group (f.pb1), ackermannia and f.pb1 mixed bacteria liquid group (a+f). Four groups of mice were each injected with 10 times quepirole (Quinpirole, QNP) for constructing compulsive disorder models, and were respectively filled with 200 μl of PBS, 200 μl of ackermann bacteria solution, 200 μl of f.pb1 bacteria solution, and 200 μl of L A +f mixed bacteria solution once daily for two weeks after modeling. The brain tissue (cortex) and colon of the mice are used for detection by a Western blot method, a qPCR method and an HPLC method. This example uses qPCR and Western blot experiments to detect expression of mouse cortical Tyrosine Hydroxylase (TH) and High Performance Liquid Chromatography (HPLC) to detect Dopamine (DA) content of the mouse cortical neurotransmitter. As a result, it was found that the levels of mRNA and protein were increased in the cerebral cortex TH of the mice of AKK, F.PB1, A+F groups, and the cortical DA content was increased, and the A+F groups exhibited the most significant difference, as compared with the PBS group (FIG. 5).

Experimental method 2: the experimental animals are randomly divided into four groups, namely PBS group (PBS), microcapsule PBS group (MC-PBS), A+F mixed bacterial liquid group (A+F) and microcapsule A+F mixed bacterial liquid group (MC-A+F). Four groups of mice were injected with Quepirole (QNP) 10 times for constructing compulsive disorder model, and 200 μl or equal amount of PBS, microcapsule PBS, a+f mixed bacterial liquid, and microcapsule a+f mixed bacterial liquid were respectively injected once daily for two weeks after modeling. The brain tissue (cortex) and colon of the mice are used for detection by a Western blot method, a qPCR method and an HPLC method. The expression of mouse cortical Tyrosine Hydroxylase (TH) was detected by qPCR and Western blot experiments, and the content of the mouse cortical neurotransmitter Dopamine (DA) was detected by High Performance Liquid Chromatography (HPLC). As se:Sub>A result, it was found that the mRNA and protein levels of the cerebral cortex TH were increased in both the A+F group and the MC-A+F group of mice, the cortical DA content was increased, and the MC-A+F group exhibited se:Sub>A more significant difference, as compared with the PBS group, and no significant difference was seen in the MC-PBS group as compared with the PBS group (FIG. 6).

Example 3 in vitro tolerance test of microencapsulated bacterial cocktail to improve forced behavior

Ackermans bacterial liquid and F.PB1 bacterial liquid grown to the plateau stage are collected, and are respectively centrifuged for 10min at 4000rpm at 4 ℃ by a refrigerated centrifuge, and the supernatant is discarded. The cells were resuspended in sterile PBS in an anaerobic incubator, centrifuged at 4000rpm at 4℃for 10min, the supernatant discarded, washed three times repeatedly and mixed. Then, the bacterial cells were resuspended in sterile PBS containing 25% (volume fraction) glycerol, and the OD600 of the bacterial solution was adjusted to 1.2, and kept at-80℃for further use.

(1) Tolerance of probiotic microcapsules in simulated gastric fluid

The Simulated Gastric Fluid (SGF) is prepared by taking 16.4mL of dilute hydrochloric acid, adding 800mL of water, adjusting pH to 2.0, adding 10g of pepsin, stirring uniformly, and adding water to a constant volume of 1000mL. Adding 0.5g microcapsule into simulated gastric fluid (4.5 mL), mixing thoroughly for 15s, shaking at 37deg.C for 30min, 60min, 90min, 120min, 150min in a shaker at 200r/min, centrifuging and sampling respectively, and calculating viable count. And a control test was performed.

(2) Release of probiotic microcapsules in simulated intestinal fluid

Simulated artificial intestinal fluid (SIF): 6.8g of monopotassium phosphate, 500mL of distilled water is added for dissolution, and 0.4wt% of sodium hydroxide is used for regulating the pH to 6.8; sterilizing at 120deg.C for 20min; 10g of trypsin, adding a proper amount of sterile water to dissolve, mixing the two solutions, and adding water to fix the volume to 1000mL. Adding 0.5g of microcapsule bacterial mixture for improving compulsory behavior into simulated intestinal juice (4.5 mL), mixing thoroughly for 15s, shaking at 37deg.C for 30min, 60min, 90min, 120min and 150min in a shaker at 200r/min, centrifuging and sampling respectively, and calculating viable count.

The survival rate of the microcapsule bacterial mixture for improving the compulsory behavior in simulated gastric fluid is shown in fig. 7, and it can be seen from fig. 7 that in simulated artificial gastric fluid (SGF), the free A+F mixed bacterial liquid is completely dead within 30min, and the microcapsule A+F mixed bacterial liquid still remains alive within 90 min. The reason for producing the results is that the microcapsule three-layer package can effectively resist gastric acid erosion and can furthest protect mixed bacterial liquid in gastric acid environment.

The survival rate of the microcapsule bacterial mixture for improving the compulsory behavior in the simulated intestinal fluid is shown in fig. 8, and as can be seen from fig. 8, in the simulated artificial intestinal fluid (SIF), the free mixed bacterial liquid of A+F rapidly dies with time, and the mixed bacterial liquid of the microcapsule A+F still remains alive within 3 hours. The reason for producing the results is that the three-layer package of the microcapsule can effectively slow down the contact time of the intestinal juice and the mixed bacterial liquid and improve the viability of the mixed bacterial liquid in simulating artificial intestinal juice.

The experiments show that the Acremonium and the F.PB1 bacteria have the functions of relieving compulsive behavior and anxiety-like behavior related to compulsive disorder, the effects probably play roles by regulating intestinal barrier, brain inflammatory factors and neurotrophic factors in intestinal-brain axes and being related to neurotransmitter functions, and the microcapsule packaging can improve the effects and is beneficial to the release of bacterial liquid in colon. The application provides a practical method for providing a powerful theoretical basis and bacterial liquid as medicine application in seeking the treatment potential of probiotics on obsessive-compulsive disorder, and has higher social significance and potential market value.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims (10)

1. The microcapsule bacterial mixture for improving the compulsive behavior is characterized in that the microcapsule bacterial mixture is of a core-shell structure, the outer layer is Arabic gum, the middle layer is chitosan, and the inner layer is a mixed solution of the bacterial mixture and sodium alginate;

the bacteria in the bacterial mixture are Acremonium and F.PB1 bacteria.

2. A method of preparing the forced behavior improving micro-encapsulated bacterial mixture of claim 1, comprising the steps of:

step 1: collecting Ackermans bacterial liquid and F.PB1 bacterial liquid which grow to a plateau, respectively centrifuging, discarding the supernatant, re-suspending the bacterial bodies by using sterile PBS, respectively centrifuging, discarding the supernatant, repeatedly washing for three times, mixing, re-suspending the bacterial bodies by using sterile PBS containing 25% glycerol, and regulating the OD600 of the bacterial liquid to be 1.2;

step 2: 2-10 mL of the bacterial liquid in the step 1 is taken and added into sodium alginate solution according to the volume ratio of 1:1, and magnetically stirred to obtain solution;

step 3: extruding the solution obtained in the step 2 into 0.1M CaCl at constant speed ₂ Forming a calcium alginate gel in the solution, hardening the gel at 4 ℃ for 1h, removing the supernatant, and washing the calcium alginate gel with a sodium chloride solution;

step 4: immersing the calcium alginate gel into a chitosan water solution, uniformly stirring to obtain calcium alginate/chitosan gel, washing the calcium alginate/chitosan gel with a NaCl solution, and standing;

step 5: immersing the calcium alginate/chitosan gel in the step 4 in a 1wt% acacia solution, stirring and coating for 30min, and filtering and recovering the microcapsule bacterial mixture for improving the compulsive behavior.

3. The method for preparing a forced behavior-improving microcapsule bacterial mixture according to claim 2, wherein an effective dose of said ackerman bacteria liquid and f.pb1 bacteria liquid is 200 μl, and a concentration of a mixed bacteria liquid of said ackerman bacteria liquid and f.pb1 bacteria liquid is 1.2x10 ⁹ CFU。

4. The method for preparing a mixture of microencapsulated bacteria for improving compulsory behavior according to claim 2, wherein in step 1, the temperature is 4 ℃ and the rotational speed is 4000rpm for 10min in both centrifugation steps.

5. The method for preparing a forced behavior improving microcapsule bacterial mixture according to claim 2, wherein in the step 2, the magnetic stirring time is 30-60 min, and the rotating speed is 200 rpm.

6. Use of the micro-encapsulated bacterial mixture for improving obsessive-compulsive behaviour as claimed in claim 1 in the manufacture of a medicament for the treatment of obsessive-compulsive disorder.

7. The use according to claim 6, wherein the obsessive-compulsive disorder includes, but is not limited to obsessive-compulsive disorder caused by immune dysfunction of the gut.

8. The use of claim 6, wherein the dosage form of the medicament comprises any one of the dosage forms that is pharmaceutically acceptable.

9. The use of claim 8, wherein the formulation of the medicament comprises any one of a liquid formulation, a solid formulation, a semi-solid formulation, and a gaseous formulation.

10. The use according to claim 6, wherein the mode of administration is intragastric administration.